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Structure, function and mechanisms of G-Proteins
Oliver Daumke
MDC-Berlin, House 31.2 (Flachbau), R0225
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1994 Nobel Prize in Medicine, Alfred Gilman and Martin Rodbell, for their „discovery of G-Proteinsand the role of these proteins in signal transduction in cells.“
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G-Protein = Guanine-nucleotide binding protein(GNBD)
12
5
43
Guanine
Ribose
Phosphates
α
1
3
42
65 7
89
Guanosine
EsterAnhydride
Guanosine-triphosphate - GTP
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G-Protein families
• Heterotrimeric G-Proteins (Transducin, Gi, Gq …), in 7-TM receptor signalling
• Initiation, elongation, termination factors in protein synthesis (IF1, EF-Tu, EF-TS)
• Signal recognition particle (SRP) and its receptor, translocation of nascent polypeptide chains in the ER
• Ras-like GTPases (Ras, Rap, Rho, Ran, Rab, Arf, Arl, Sar), molecular switches in signal transduction
• Dynamin superfamily of GTPases, remodelling of membranes
+ 60 further distinct families
Leipe et al., JMB (2002)
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The G-domain
Mixed - protein
5 conserved motifs (G1-G5) involved in nucleotide binding
Pai et al., Nature (1989)
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Ras-like G-Proteins are molecular switches
Effector: Interacts stably with the GTP-bound form GEF: Guanine nucleotide Exchange FactorGAP: GTPase Activating Protein
To allow switch function: highaffinity for nucleotide required pMol
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Vetter and Wittinghofer, Science (2001)
The switch regions
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The GTPase reaction
• Intrinsic GTPase rates of small G-Proteins are slow (range: kcat=10-2 - 10-3 min-1)
• SN2 nucleophilic attack with trigonal bipyramidal transition state
• Phosphate hydrolysis reaction is thermodynamically highly favourable but kinetically very slow (Westheimer FH
(1987), Why nature chose phosphates, Science 235, 1173-1178)
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Enzymatic strategies for GTP hydrolysis
1) Counteracting of negative charge at phosphates
- P-loop (GxxxxGKS), hydrogen bonds and lysine
- Mg2+ ion, essential for nucleotide binding and hydrolysis
- catalytic arginine (and lysine residues)
2) Positioning of attacking nucleophile
- catalytic glutamine
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Non-hydrolysable GTP analogues
Abbreviations
GTP--S
GMPPCP
GMPPNP
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Transition state mimicks of GTP hydrolysis
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GTPase Activating Proteins
• Accelerate intrinsic GTPase by a factor of 105 – 106
• Ras, Rap, Rho, Rab, Ran have completely unrelated GAPs
• High affinity binding to the GTP-bound form, low affinity interaction with the GDP-bound form
• Mechanism of GTP hydrolysis ?
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Monitoring the GAP-catalysed reaction
G-Protein (GTP) + GAP
G-Protein (GTP)GAP
G-Protein (GDP) Pi GAP
G-Protein (GDP) GAP
G-Protein (GDP) + GAP
k1 k2
k3
k4
k5
Pi
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Multiple-turnover assays
• Monitors several rounds of GAP catalysed G-Protein (GTP) hydrolysis
• G-Protein (GTP) as substrate, in excess, e.g. 200 µM• GAP in catalytic amounts, e.g. 100 nM• Determine initial rates of GTP hydrolysis by
– HPLC (ratio GDP, GTP)– Thin layer chromatography using radioactively
labelled GTP– Phosphate release (colorimetric assay, radioactive
assays)• Vary concentration of G-Protein to determine
Michaelis-Menten parameters (KM, kcat)
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Monitoring the GAP-catalysed reaction
G-Protein (GTP) + GAP
G-Protein (GTP)GAP
G-Protein (GDP) Pi GAP
G-Protein (GDP) GAP
G-Protein (GDP) + GAP
k1 k2
k3
k4
k5
Pi
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Single-turnover assays
• Analysis of a single cycle of GTP hydrolysis• Often monitored by fluorescence stopped-flow• Typically 1 – 2 µM fluorescently labelled G-Protein (GTP)
in one cell, excess of GAP in the other cell• Vary concentration of GAP → multiparameter fit allows
determination of k1, k2, KD, …
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The mechanism of RasGAP
Scheffzek et al., Nature (1996)
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Fluorescence increase:
complex formation
Fluorescence decrease:
GTP hydrolysis
Fluorescence stopped-flow to monitor the GAP reaction
Ras(mantGTP) vs. RasGAP
Ahmadian et al., Nature Structure Biology (1997)
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Ras(mantGTP) vs. RasGAP
An arginine residue in RasGAPs is essential for GAP activity
Ahmadian et al., Nature Structure Biology (1997)
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AlF3 promotes formation of a transition state complex
Mittal et al., Science (1994)
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Scheffzek et al., Science (1997)
The RasGAP-Ras complex
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• Involved in various signalling pathways, e.g. integrin activation
• close Ras homologue BUT: No catalytic glutamine residue
• own set of GAPs with no sequence homology to RasGAPs
Rap1
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0 100 200 300 400 5000
20000
40000
60000
80000
100000
120000
140000
160000
180000c
ou
nts
sec
100 nM RapGAP
800 µM Rap1(GTP)
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Rap1GAP stimulates intrinsic Rap1 reaction 100.000 fold
kcat= 6 s-1
Km = 50 µM
Brinkmann et al., JBC (2001)
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No arginine finger is involved in catalysis
Brinkmann et al, JBC (2001)
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The Rap1GAP Dimer
Daumke et al., Nature (2004)
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The catalytic domain of Rap1GAP has a G-domain fold
Ras
Rap1GAP cat
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Rap1-Rap1GAP reaction followed by fluorescence stopped-flow
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R286 is not essential for the GAP reaction
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His287 is involved in binding to Rap1
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Rap1GAP provides a catalytic Asn, the „Asn thumb“, for catalysis
Daumke et al., Nature (2004)
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Asn290 is a purely catalytic residue and not involved in binding to Rap1
Kd = 4 M
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Rap1GAP-Rap1 complex indicates that Asn thumb positions attacking water molecule
Scrima et al., EMBOJ (2008)
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The Dynamin-family of GTPases
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The shibire fly
Bing Zhang, UT Austin
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Wt 30°C Drosophila nerve terminalKosaka and Ikeda, J Neurobiol., 1982
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shibire 30°C Drosophila nerve terminalKosaka and Ikeda, J Neurobiol., 1982
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The family of Dynamin-related GTPases
• Classical Dynamins: Dyn1, Dyn2, Dyn3
• Dynamin-related proteins: Mx, Mitofusin• GBP-related proteins: GBPs, Atlastins• Bacterial Dynamins
GTPase Middle PH GED PRD
Common features:- Low affinity for nucleotide- Template induced self-oligomerisation- Assembly-stimulated GTP hydrolysis
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1000 x stimulation of Dynamin‘s GTPase reaction by lipid tubule binding
Stowell et al., Nat Cell Biol (1999)
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What is the mechanism of Dynamin ?
Sever et al., Nature (1999)N&V by T. Kirchhausen
Constrictase Effector
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Stowell et al., Nat Cell Biol (1999)www.endocytosis.org
No Dynamin GTP--S GDP
Is Dynamin a popase ?
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Is Dynamin working as a twistase ?
Roux et al., Nature (2006)Dynamin, no nucleotide
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Roux et al., Nature (2006)Dynamin, addition GTP
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Roux et al., Nature (2006)Dynamin, addition GTP
Biotin-Dynaminstreptavidin – polysterene bead
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• EHD = Eps15 homology domain containing protein
• Highly conserved in all higher eukaryotes, but not in yeast and bacteria
• Four paralogues in human, 70 - 80% amino acid identity
The EHD family
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Biochemical features
• Binds to adenine and not guanine nucleotides with affinity in the low micromolar range
• Binds to negatively charged liposomes• Liposome-stimulated ATP hydrolysis (very slow)
PS liposomes
+ EHD2
Daumke et al., Nature (2007)
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Daumke et al., Nature (2007)
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Lipid binding site of EHD2
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Implications for membrane remodelling
Factors involved in membrane remodelling / destabilisation• Oligomer formation into rings around a lipid template• Insertion of hydrophobic residues into outer membrane bilayer• Interaction of highly curved membrane interaction site
perpendicular to curvature of lipid tubule• Conformational changes upon ATP hydrolysis
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Acknowledgements / References
• Alfred WittinghoferVetter and Wittinghofer „The Guanine nucleotide binding switch in three
dimensions.“ Science (2001)Bos, Rehmann, Wittinghofer „GEFs and GAPs critical elements in the
control of G-Proteins.“ Cell (2007)A. Wittinghofer, H. Waldmann. „Ras - A molecular switch involved in tumor
formation.“ Angew. Chem. Int. Ed. (2000)Scheffzek, Ahmadian, Kabsch, Wiesmuller, Lautwein, Schmitz &
Wittinghofer „The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants.” Science (1997)
• Harvey McMahon (www.endocytosis.org)Praefcke, McMahon, „The dynamin superfamily: universal membrane
tubulation and fission molecules?” Nat Rev Mol Cell Biology (2004)McMahon, Gallop, „Membrane curvature and mechanisms of dynamic cell
membrane remodelling”, Nature (2005)